The most common anomaly of the genitourinary system is hypospadias, a malformation of the external genitalia that is characterized by failure of urethral tube closure and incomplete formation of the prepuce (foreskin) and ventral penis. The incidence of hypospadias has more than doubled over the past 30 years, without explanation, and it is now the second most common birth defect in humans, affecting an estimated 1 of every 250 live births. Whether the causes of hypospadias are genetic, environmental, or a combination of both, development of preventative strategies for hypospadias and of cell-based therapies for repair of urologic tissues will require knowledge of the genetic programs that direct cell fate decisions and tissue morphogenesis. Identification of these mechanisms first requires the establishment of a foundation of descriptive data, in the form of gene expression patterns, for the different tissue compartments of the lower genitourinary tract. Moreover, transcriptional profiling will need to be linked to developmental anatomy in order to formulate hypotheses regarding gene function during morphogenesis, and to consider how multiple genes may interact within a tissue or cell population. In preliminary studies using microarrays, we identifie 86 novel markers of the urethral epithelium of the El 2.5 genital tubercle. Here we propose to use OPT to build a 3D reference model of the genital tubercle, and to map the expression domains of each of these genes onto the reference tubercle to create a 3D, multi-gene atlas of the gene expression in the developing external genitalia. We will build upon these results in two ways. Firstly, we will extend the OPT atlas to two additional stages of external genital development. Secondly, we will drill deeper into the expression profiling by using NexGen sequencing technology (RNA-seq) to characterize the quantitative patterns of gene expression and the distribution of splice variants in the endodermal cells fated to form the urethral epithelium. This combination of approaches will provide a wealth of new cell type-specific markers, uncover the quantitative molecular profiles of urethral progenitors, and place these data in a 3D developmental anatomical context that can be utilized by the research community for future hypothesis-based studies.